Original Article

Orthodontic appliances did not increase risk of dental caries and

periodontal disease under preventive protocol
Ana Zilda Nazar Bergamoa; Katharina Morant Holanda de Oliveiraa; Mı́rian Aiko Nakane
Matsumotob; Cássio do Nascimentoc; Fábio Lourenço Romanob; Raquel Assed Bezerra da Silvab;
Lea Assed Bezerra da Silvab; Paulo Nelson-Filhob

ABSTRACT
Objectives: To assess periodontal parameters and microbial species levels after orthodontic
appliance placement in patients who received oral hygiene instructions and who were monitored
and motivated throughout the study.
Materials and Methods: The Periodontal Index was recorded and saliva collection was performed
before (T0) and 30 (T1), 60 (T2), and 90 (T3) days after orthodontic appliance placement in 15
patients (mean age 17.53 6 8.0 years). Analysis was carried out using checkerboard DNA-DNA
hybridization. Nonparametric statistical analysis was performed.
Results: The Periodontal Index did not change. The total amount of the purple and red complexes
and Candida species showed a significant decrease from T2. The green, yellow, and orange
complex showed a significant decrease at T3. The specific species analysis showed that Prevotella
nigrescens, Pseudomonas putida, Fusobacterium periodonticum, Pseudomonas aeruginosa,
Peptostreptococcus anaerobius, and Tanerella forsythia showed high incidence before bonding,
and their levels decreased at T2 and T3. Only Porphyromonas gingivalis showed increased levels
at T2 and displayed the highest level at T3. The Streptococcus group decreased their levels from
T2 onward.
Conclusions: A dynamic change in microbial levels was identified. The decrease in the levels of
complexes present was only possible due to the mechanical method of oral hygiene implemented
in this sample. (Angle Orthod. 2019;89:25–32.)
KEY WORDS: Periodontal microbiology; Orthodontic brackets; Bacteria; Checkerboard DNA-DNA
hybridization; Oral hygiene

INTRODUCTION tic interactions among the microorganisms contribute

Microbes are present in both healthy and diseased
environments. Synergistic, mutualistic, and antagonis-
a
PhD Student, Department of Pediatric Clinic, School of
Dentistry of Ribeirão Preto, University of São Paulo, São Paulo,
Brazil.
b
Professor, Department of Pediatric Clinic, School of Dentist-
ry of Ribeirão Preto, University of São Paulo, São Paulo, Brazil.
c
Professor, Department of Dental Materials and Prosthodon-
tics, School of Dentistry of Ribeirão Preto, University of São
Paulo, São Paulo, Brazil.
Corresponding author: Dr Ana Zilda Nazar Bergamo, Depart-
ment of Pediatric Clinic, School of Dentistry of Ribeirão Preto,
University of São Paulo, Avenida do Café, S/N, Monte Alegre,
CEP: 14040-904, Ribeirão Preto, SP, Brazil
(e-mail: anaznbergamo@gmail.com)
Accepted: July 2018. Submitted: February 2018.
Published Online: September 21, 2018
Ó 2019 by The EH Angle Education and Research Foundation,
Inc.
to the development of a polymicrobial biofilm.1,2 Initial
biofilm formation plays an important role in under-
standing the growth and proliferation of microorgan-
isms.3–6
The literature describes that 60% of all orthodontic
patients experience some alteration in biofilm accumu-
lation after the bonding of orthodontic appliances.
Authors emphasize a need for preventive measures to
prevent biofilm-related complications during orthodon-
tic treatment. Knowledge of the microbial dynamics in
the early stages of orthodontic treatment can assist in
the adoption of effective measures to prevent these
changes.7
Studies investigating microbial contamination during
orthodontic treatment have evaluated the species that
are related to dental caries8–11 or periodontal dis-
ease.11,12 Few studies have evaluated the microbial

DOI: 10.2319/022118-139.1 25 Angle Orthodontist, Vol 89, No 1, 2019

26 BERGAMO, DE OLIVEIRA, MATSUMOTO, NASCIMENTO, ROMANO, DA SILVA, DA SILVA, NELSON-FILHO
ecology in the oral cavity after the bonding of
orthodontic appliances.13,14
According to Socransky and Haffajee,15 there are
specific associations among bacterial species in the
supragingival biofilm. Based on this relationship, they
are grouped into different complexes. Purple and green
complex species are present in early biofilm formation
because of the distinct patterns of coaggregation. Yellow
complex returns very quickly after cleaning surfaces of
the teeth; this is an intermediate complex that improves
the attachment points of adhesion. The orange and red
complexes are directly involved with periodontal disease
and are frequently associated with the soft tissue
inflammation observed during orthodontic treatment.16,17
The aim of this clinical study was to assess periodontal
parameters and to examine microbial communities in
saliva in the early stages of orthodontic treatment in
patients who received oral hygiene instructions, moni-
toring, and motivation throughout the study.
MATERIALS AND METHODS
The Institutional Research Ethics Committee grant-
ed approval for the research project (Process
0062.0.138.000-10). Fifteen patients of both sexes
(one male and 14 females) aged 11 to 41 years (mean
age ¼ 17.53 6 8.0 years) with permanent dentition
were screened.
The following inclusion criteria were considered in
this study: no previous orthodontic treatment; no use of
antibiotics, antimicrobial mouthwashes, or any system-
ic medication within 3 months prior to the study; no
periodontal treatment within the previous 3 months; no
smoking; no clinical signs of gingivitis; and no systemic
disorder that could alter the periodontal conditions prior
to bracket bonding.
The plaque index (PI) and gingival index (GI) were
measured at three sites per tooth (mesiobuccal ¼ MB,
buccal ¼ B, and distobuccal ¼ DB),18 and gingival
bleeding index (GBI)19 using a PCPUNC-BR15 probe
(HuFriedy of Brazil, Rio de Janeiro, RJ, Brazil) was
determined to the nearest millimeter. These indices were
measured before bonding (T0) and 30 (T1), 60 (T2), and
90 (T3) days after bonding in the upper and lower arches.
Standardized hygiene instructions (modified Bass
brushing technique) were given to all patients by the
same investigator. The subjects were asked to brush
three times daily, after meals, and were instructed not
to use any hygiene products other than toothpaste and
dental floss. Recall visits were scheduled at 30 days, at
which time the instructions were reinforced.
The patients received edgewise metallic orthodontic
brackets (0.022 3 0.028-inch slot) (Dental Morelli,
Sorocaba, SP, Brazil) in the upper and lower arches.
Angle Orthodontist, Vol 89, No 1, 2019
The brackets were bonded with orthodontic light-cured
adhesive (Transbond XT, 3M Unitek, Monrovia, Calif).
Saliva Collection
Nonstimulated saliva (1 mL) was collected in Falcone
Conical Centrifuge tubes (Thermo Scientifice Nunce -
Waltham, MA USA) at T0, T1, T2, and T3. The saliva
was collected in the morning. After collection, the Falcon
tube was centrifuged for 30 seconds, and 30 lL of saliva
was transferred to an Eppendorf tube (Eppendorf AG,
Hamburg, Germany) with a content of 120 lL of buffer
solution (10 mM Tris-HCL [Sigma-Aldrich Co., St Louis,
Mo]), pH 7.6). Following this, 100 lL of NaOH (Labsynth
Product Laboratories, Diaderma, SP, Brazil) was added.
The samples were stored at 208C until the Checker-
board DNA-DNA hybridization analysis was performed
according to the method of Bergamo et al.20
Checkerboard DNA-DNA Hybridization
The levels of five Candida species and 38 bacterial
species were analyzed (Table 1). After thawing, the
samples were boiled for 5 minutes. After cooling, 800
lL of 5 M ammonium acetate was added, and the
contents were applied to the extended slot in the
MiniSlot apparatus (Immunetics Inc, Boston, Mass)
and then concentrated onto a 15 3 15-cm nylon
membrane (Yond N, Amershan Biosciences, Bucking-
hamshire, UK), followed by baking for 2 hours at 808C.
Control samples defined amounts of genomic DNA
corresponding to either 105 or 106.
The membranes were prehybridized (buffer hybrid-
ization; NaCl 0.5 M; blocking reagent 0.4% [w/v]). After
prehybridization, the membranes were placed in a
Miniblotter 45 (Immunetics). Defined amounts of
fluorescein-labeled whole genomic probes were diluted
in 150 mL of hybridization solution, applied in individual
lanes of the Miniblotter, and the whole apparatus was
placed in a sealed plastic bag containing sheets of
wetted paper towel. Hybridization was performed
overnight at 608C with gentle agitation. The following
day, the membranes were washed twice in a solution
of 2 M urea, 0.1% sodium dodecyl sulfate, 50 mM
NaH2PO4 (pH 7.0), 150 mM NaCl, 1 mM MgCl2, and 0.2
blocking reagent at 658C for 30 minutes and were also
washed twice in a solution of 1 M Tris base, 2 M NaCl,
and 1 M MgCl2 for 15 minutes at room temperature.
The hybrids were detected by chemiluminescence
using the Gene Images CDP-Star detection module
(GE Healthcare, Buckingham, UK). Chemiluminescent
signals were detected by exposing the membrane to
ECL Hyperfilm MP (GE Healthcare) for 10 minutes.
The image obtained on the hyperfilm was digitized and
analyzed by the TotalLabe Quant v13 software
(TotalLab Ltd, Newcastle, UK).
HYGIENE PROTOCOL SUSTAINS BACTERIAL LEVELS 27

Table 1. Microbial Count (lg 3 105) in the Saliva, Before Bonding Brackets and 30, 60, and 90 Days After Bonding Bracketsa
Microorganism (ATCC No.) T0 M(Q1-Q3) T1 M(Q1-Q3) T2 M(Q1-Q3) T3 M(Q1-Q3) P Friedman
Purple complex
Veillonella parvula (10790) 5.17 (4.34–6.14) 5.16 (4.81–5.54 3.45 (2.94–3.94) 3.70 (3.09–4.05) .00001*
Neisseria mucosa (25996) 3.01 (0.00–3.29) 4.18 (3.954.43) 0.00 (0.00–0.00) 0.00 (0.000.00) .00001*
Green complex
Capnocytophaga gingivalis (33624) 0.00 (0.00–0.00) 0.00 (0.00–0.00) 0.00 (0.00–0.00) 0.00 (0.00–0.00) .112
Eikenella corrodens (23834) 3.55 (3.37–3.82) 3.96 (3.86–4.02) 2.69 (0.00–2.75) 0.00 (0.00–0.00) .00001*
Aggregatibacter a a (29523) 3.01 (0.00–3.39) 2.90 (0.00–3.03) 0.00 (0.00–3.37) 0.00 (0.00–2.73) .066
Aggregatibacter a b (29522) 2.96 (0.00–3.27) 2.81 (0.00–2.94) 3.04 (0.00–3.52) 0.00 (0.00–0.00) .021*
Yellow complex
Streptococcus mitis (49456) 3.84 (3.77–4.07) 3.88 (3.41–3.99) 4.51 (4.13–6.37) 3.57 (0.00–4.52) .013*
Streptococcus gordonii (10558) 3.82 (3.55–4.32) 3.99 (3.74–4.61) 0.00 (0.00–3.73) 3.31 (0.00–3.80) .00001*
Streptococcus constelatus (27823) 3.22 (0.00–3.57) 0.00 (0.00–3.43) 3.41 (2.82–3.99) 0.00 (0.00–3.33) .014*
Streptococcus oralis (35037) 5.23 (4.35–6.61) 5.21 (4.60–5.80) 4.04 (0.00–5.38) 0.00 (0.00–3.99) .00001*
Streptococcus sanguinis (10556) 5.73 (4.61–7.10) 5.07 (4.62–6.01) 2.76 (0.00–3.70) 0.00 (0.00–3.34) .00001*
Orange complex
Campylobacter rectus (33238) 0.00 (0.00–0.00) 0.00 (0.00–0.00) 0.00 (0.00–0.00) 0.00 (0.00–0.00) .733
Fusobacterium nucleatum (25586) 3.91 (3.54–4.08) 4.02 (3.86–4.27) 0.00 (0.00–0.00) 0.00 (0.00–0.00) .00001*
Fusobacterium periodonticum (33693) 4.24 (3.98–4.75) 3.99 (3.88–4.18) 0.00 (0.00–2.72) 0.00 (0.00–0.00) .00001*
Prevotella intermedia (25611) 0.00 (0.00–0.00) 0.00 (0.00–0.00) 5.47 (3.73–7.41) 3.74 (3.07–5.34) .00001*
Prevotella melaninogenica (25845) 0.00 (0.00–0.00) 0.00 (0.00–0.00) 5.20 (3.59–5.97) 0.00 (0.00–3.45) .00001*
Prevotella nigrescens (25261) 5.05 (4.29–6.12) 5.17 (4.54–5.70) 0.00 (0.00–0.00) 0.00 (0.00–2.89) .00001*
Parvimonas micra (33270) 0.00 (0.00–0.00) 0.00 (0.00–0.00) 0.00 (0.00–2.89) 0.00 (0.00–0.00) .003*
Red complex
Treponema denticola (35405) 4.02 (3.77–4.34) 3.83 (3.70–3.97) 0.00 (0.00–2.79) 0.00 (0.00–0.00) .00001*
Tanerella forsythia (43037) 5.38 (4.44–6.00) 4.29 (4.73–5.15) 2.97 (0.00–3.56) 0.00 (0.00–0.00) .00001*
Porphyromonas gingivalis (33277) 0.00 (0.00–0.00) 0.00 (0.00–0.00) 3.38 (0.00–4.22) 3.68 (2.81–4.86) .00001*
Cariogenic
Lactobacilos casei (393) 0.00 (0.00–0.00) 0.00 (0.00–0.00) 3.71 (2.74–3.92) 0.00 (0.00–3.78) .00001*
Streptococcus mutans (25175) 4.23 (3.77–4.57) 3.82 (0.00–4.09) 3.16 (0.00–4.16) 3.67 (3.32–4.56) .019*
Streptococcus sobrinus (27352) 6.83 (4.87–9.85) 5.58 (3.98–6.49) 3.77 (2.84–4.47) 0.00 (0.00–2.76) .00001*
Other species
Bacteroides fragilis (25285) 0.00 (0.00–0.00 0.00 (0.00–0.00) 3.24 (0.00–3.72) 0.00 (0.00–0.00) .00001*
Escherichia coli (10798) 0.00 (0.00–0.00) 0.00 (0.00–0.00) 0.00 (0.00–0.00) 0.00 (0.00–0.00) .300
Enterococcus faecalis (51299) 0.00 (0.00–0.00) 0.00 (0.00–3.05) 0.00 (0.00–0.00) 0.00 (0.00–0.00) .352
Klebsiella pneumoniae (700603) 0.00 (0.00–0.00) 0.00 (0.00–0.00) 0.00 (0.00–3.04) 0.00 (0.00–3.72) .001*
Mycoplasma salivarium (23064) 0.00 (0.00–0.00) 0.00 (0.00–0.00) 0.00 (0.00–0.00) 0.00 (0.00–0.00) .543
Pseudomonas aeruginosa (27853) 4.16 (4.09–4.29) 4.09 (3.97–4.18) 0.00 (0.00–0.00) 0.00 (0.00–0.00) .00001*
Peptostreptococcus anaerobius (27337) 5.51 (5.08–5.61) 5.16 (4.90–5.37) 0.00 (0.00–0.00) 0.00 (0.00–0.00) .00001*
Pseudomonas putida (12633) 5.70 (4.84–6.33) 5.25 (4.73–6.24) 0.00 (0.00–3.51) 0.00 (0.00–0.00) .00001*
Porphyromonas endodontalis (35406) 0.00 (0.00–0.00) 0.00 (0.00–0.00) 2.76 (0.00–3.02) 0.00 (0.00–0.00) .001*
Staphylococcus aureus (25923) 0.00 (0.00–3.40) 0.00 (0.00–3.01) 0.00 (0.00–0.00) 0.00 (0.00–3.40) .093
Solobacterium moreei (CCUG39336) 5.17 (4.28–5.53 4.55 (4.34–5.24) 5.40 (3.58–6.38) 0.00 (0.00–3.68) .00001*
Streptococcus parasanguinis (15911) 6.33 (4.62–7.63) 5.07 (4.60–5.66) 3.76 (0.00–4.61) 0.00 (0.00–0.00) .00001*
Staphylococcus pasteuri (51129) 4.49 (4.12–4.85) 3.95 (3.47–4.36) 0.00 (0.00–3.71) 3.85 (2.21–4.81) .005*
Streptococcus salivarius (25975) 5.18 (4.57–6.64) 4.97 (4.59–5.75) 4.31 (3.26–5.92) 3.04 (0.00–4.02) .00001*
Candidas
Candida tropicalis (13803) 0.00 (0.00–3.44) 0.00 (0.00–0.00) 0.00 (0.00–0.00) 0.00 (0.00–0.00) .055
Candida albicans (10231) 4.87 (3.90–5.31) 4.64 (4.08–4.96) 4.24 (3.45–5.00) 0.00 (0.00–2.94) .00001*
Candida dubliniensis (44508) 4.45 (4.04–4.70) 4.02 (3.95–4.09) 0.00 (0.00–0.00) 0.00 (0.00–0.00) .00001*
Candida glabrata (66032) 5.41 (4.70–6.05) 6.08 (5.00–6.42) 0.00 (0.00–0.00) 0.00 (0.00–0.00) .00001*
Candida krusei (2159) 4.90 (4.4–6.64) 5.79 (5.15–6.84) 0.00 (0.00–0.00) 0.00 (0.00–0.00) .00001*
a
ATCC indicates American Type Culture Collection; T0, saliva sample before bonding; T1, saliva sample 30 days after bonding; T2, saliva
sample 60 days after bonding; T3, saliva sample 90 days after bonding; M, median; Q1, first quartile; and Q3, third quartile.
* Statistically significant difference.

Statistical Analyses RESULTS

The Friedman’s nonparametric test was employed.
Multiple comparisons were achieved using the Dunn
posttest. Differences were considered significant when
P , .05. SPSS 21.0.0 statistical software (SPSS Inc,
Chicago, Ill) was used for data analysis.
No statistically significant difference was observed in
PI, GI, and GBI scores throughout the study. Figure 1
shows the median, first, and third quartiles of these
indexes for all evaluated periods.

Angle Orthodontist, Vol 89, No 1, 2019

28 BERGAMO, DE OLIVEIRA, MATSUMOTO, NASCIMENTO, ROMANO, DA SILVA, DA SILVA, NELSON-FILHO
Figure 1. Periodontal Index distribution. T0 ¼ Before bonding; T1 ¼ 30 days after bonding; T2 ¼ 60 days after bonding; T3 ¼ 90 days after bonding
of the orthodontic appliance. PI indicates plaque index; GBI, gingival bleeding index; and GI, gingival index.
All microbial complexes showed significant changes
over the study period (Friedman test P ¼ .00001), with
a decrease in the levels of the microorganisms
throughout the evaluated observational period (Figure
2). Purple and green complexes decreased levels over
the observational period. A significant decrease of the
purple started from T2, and the lowest level of the
purple complex was also identified at T2. Green
complex showed its lowest level at T3. The yellow
and orange complex levels decreased significantly
only at T3. The red complex and Candida spp
decreased significantly at T2, and the lowest value
was observed at T3 (Table 2).
Regarding individual analysis of the microorganisms,
only eight species’ levels did not change throughout
the study (Table 1). There was no positive test
observed for anaerobic species such as Prevotella
melaninogenica, Parvimonas micra, and Capnocyto-
phaga gingivalis. Some opportunistic species (Bacte-
roides fragilis, Escherichia coli) were not observed at
T0. After bonding, significant changes were observed
for P melaninogenica (T2-T0, T2-T1, and T2-T3; P ¼
.00001), P micra (T2-T0 and T2-T1; P ¼ .034), and B
fragilis (T2-T0 and T2-T1; P ¼ .007: T2-T3; P ¼ .048),
which had their highest levels identified at T2 and
which decreased significantly at T3. Out of 15 species
related to deep pockets and the status of advanced
Angle Orthodontist, Vol 89, No 1, 2019
periodontitis, eight showed significant decreases in
levels at T2 and T3: A.a.b, Prevotella nigrescens,
Pseudomonas putida, Fusobacterium periodonticum,
Pseudomonas aeruginosa, Peptostreptococcus anae-
robius, Treponema dentı́cola, and Tanerella forsythia
(Table 3). Only P gingivalis increased at T2, and its
highest level occurred at T3 (T2-T0 [P ¼ .048]; T2-T1 [P
¼ .020]; T3-T0 [P ¼ .001]; T3-T1 [P ¼ .00001]).
Among nine species of the Streptococcus group
analyzed, six species showed significantly decreased
levels throughout the study: S sobrinus, S mutans, S
sanguinis, S salivarius, S parasanguinis, and S oralis
(Table 4). Lactobacilos casei increased significantly at
T2 (T2-T0; P ¼ .002: T2-T1; P ¼ .00001). The levels
decreased significantly at T3 (T3-T2; P ¼ .011).
Candida species C albicans, C dubliniensis, C
glabrata, and C krusei showed a significant alteration
in their levels (P ¼ .00001). However, the behavior of C
albicans was different from that of the others: its levels
decreased significantly only at T3, while the others
showed a decrease starting from T2 (Table 1).
DISCUSSION
Previous literature1,2,21–23 emphasized the importance
of knowing the relationship between microbial species
and the complex oral cavity environment for the early
HYGIENE PROTOCOL SUSTAINS BACTERIAL LEVELS 29

Figure 2. Bar chart. The total counts of different microbial complex levels (lg 3 105). T0 ¼ Before bonding; T1 ¼ 30 days after bonding; T2 ¼ 60
days after bonding; and T3 ¼ 90 days after bonding of the orthodontic appliance.

diagnosis of dental caries and periodontal disease.
When the balance of the oral environment was broken,
a dynamic fluctuation of microbial levels was identi-
fied.24–26 Thus, the increase in levels of individual
species should be viewed with caution in the ortho-
dontic appliance environment.27
In this study, immediately after installation of the
orthodontic appliance, there was an increase in the
levels of purple and yellow complexes and Candida
spp (T1). At T2, levels were mildly reduced, and at T3,
the lowest levels were identified, indicating that
homeostasis was recovered.
Some periodontal pathogenic species, such as P
melaninogenica and P intermedia, were not identified
at the baseline but showed an increase in levels at T2.
Some anaerobic species have been shown26–29 to be
Table 3. Periodontal Pathogens in Which Levels Decreaseda

Table 2. Post Hoc Dunn’s Testa Microorganism Decreased at T2 Decreased at T3

Complexes T0 T1 T2 T3 A.a.b T3-T0 (P ¼ .028)

T3-T2 (P ¼ .016)
Purple 7.37 9.39 3.45* 3.76* F periodonticum T2-T0 (P ¼ .00001) T3-T0 (P ¼ .00001)
Green 9.76 9.50 3.58 0.00* T2-T1 (P ¼ .002) T3-T1 (P ¼ .001)
Yellow 20.39 20.78 15.65 9.28* P nigrescens T2-T0 (P ¼ .00001) T3-T0 (P ¼ .00001)
Orange 14.20 13.14 10.80 3.74* T2-T1 (P ¼ .00001) T3-T1 (P ¼ .00001)
Red 9.40 8.56 4.22* 3.68* T denticola T2-T0 (P ¼ .00001) T3-T0 (P ¼ .00001)
Candida spp 20.33 20.95 4.26* 0.00* T2-T1 (P ¼ .003) T3-T1 (P ¼ .00001)
Note: Median of Complexes Levels total count (lg 3 105) T forsythia T2-T0 (P ¼ .00001) T3-T0 (P ¼ .00001)
a
T0 indicates saliva sample before bonding; T1, saliva sample 30 T2-T1 (P ¼ .004) T3-T1 (P ¼ .00001)
days after bonding; T2, saliva sample 60 days after bonding; and T3, P aeruginosa T2-T0 (P ¼ .00001) T3-T0 (P ¼ .00001)
saliva sample 90 days after bonding. T2-T1 (P ¼ .001) T3-T1 (P ¼ .00001)
* Statistically significance difference: Purple complex: T0-T2, P ¼ P anaerobius T2-T0 (P ¼ .00001) T3-T0 (P ¼ .00001)
.002; T0-T3, P ¼ .003; T1-T2, P ¼ .00001; T1-T3, P ¼ .00001; Green T2-T1 (P ¼ .001) T3-T1 (P ¼ .00001)
complex: T0-T3, P ¼ .00001; T1-T3, P ¼ .00001; T2-T3, P ¼ .013;
P putida T2-T0 (P ¼ .00001) T3-T0 (P ¼ .00001)
Yellow complex: T0-T3, P ¼ .00001; T1-T3, P ¼ .004; Orange
complex: T0-T3, P ¼ .00001; T1-T3, P ¼ .00001; T2-T3, P ¼ .001; Red T2-T1 (P ¼ .00001) T3-T1 (P ¼ .00001)
complex: T0-T2, P ¼ .001; T0-T3, P ¼ .00001; T1-T2, P ¼ .04; T1-T3, a
P ¼ post hoc Dunn’s test. T0 indicates saliva sample before
P ¼ .002; Candida spp.: T0-T2, P ¼ .011; T0-T3, P ¼ .00001; T1-T2, P bonding; T1, saliva sample 30 days after bonding; T2, saliva sample
¼ .016; T1-T3, P ¼ .00001. 60 days after bonding; and T3, saliva sample 90 days after bonding.

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30 BERGAMO, DE OLIVEIRA, MATSUMOTO, NASCIMENTO, ROMANO, DA SILVA, DA SILVA, NELSON-FILHO

Table 4. Median Values (lg 3 105)—Streptococcus Group Post program implemented in the study was successful in
Hoc Dunn’s Testa reducing the levels, and control was obtained.
Microorganism T0 T1 T2 T3 S sanguinis is considered a beneficial bacterium with
Yellow complex regard to dental caries because it is an antagonist to S
S mitis 3.84 3.88 4.51 3.57 mutans. Epidemiological studies31,33 of dental caries
S gordonii 3.82 3.99 0.00* 3.31* demonstrated that early colonization and high levels of
S constelatus 3.22 0.00 3.40* 0.00
S sanguinis in a patient’s oral cavity correlated with
S oralis 5.23 5.21 4.04 0.00*
S sanguinis 5.73 5.07 2.76* 0.00* significantly delayed colonization by S mutans. The
Cariogenic species current results were in agreement with those studies,
S mutans 4.23* 3.82 3.16 3.67 since the highest levels of S sanguinis were observed
S sobrinus 6.83 5.58 3.77 0.00* at T0, decreased significantly at T2, and reached their
Others species
lowest levels at T3, when the S mutans levels had a
S parasanguinis 6.33 5.07 3.76* 0.00*
S salivarius 5.18 4.97 4.31 3.04* mild increase. This result highlighted the antagonism
between species and emphasized the predisposition of
a
T0 indicates saliva sample before bonding; T1, saliva sample 30
days after bonding; T2, saliva sample 60 days after bonding; and T3, orthodontic patients to dental caries even after
saliva sample 90 days after bonding. instruction, motivation, and supervision of oral hygiene
* Statistically significant difference: . mitis: T0-T2, P ¼ .016; T1-T2, throughout the study period.
P ¼ .006; T2-T3, P ¼ .01; S gordonii: T0-T2, P ¼ .016; T0-T3, P ¼
.040; T1-T2, P ¼ .001; T1- T3, P ¼ .002; S constelatus: T1-T2, P ¼ The L casei cariogenic species correlated with deep
0.020; T2-T3, P ¼ .009; S oralis: T0-T2, P ¼ .00001; T0-T3, P ¼ cavities and increased significantly at T2, but de-
.00001; T1-T2, P ¼ .004; T1-T3, P ¼ .001; S sanguinis: T0-T2, P ¼ creased significantly at T3, indicating a return to
.00001; T0-T3, P ¼ .00001; T1-T2, P ¼ .004; T1-T3, P ¼ .001; S
mutnas: T0-T1, P ¼ .19; T0-T2, P ¼ .010; S sobrinus: T0-T2, P ¼ .001; baseline levels. This species is correlated with carious
T0-T3, P ¼ .00001; T1-T2, P ¼ .048; T1-T3, P ¼ .00001; S dentin,32 and the sample did not show cavity activity.
parasanguinis: T0-T2, P ¼ .001; T0-T3, P ¼ .00001; T1-T3, P ¼ Candida spp are frequently correlated with a
.00001; T2-T3, P ¼ .048; S salivarius: T0-T3, P ¼ .00001; T1-T3, P ¼
.00001; T2-T3, P ¼ .016. decrease of pH, increase of orthodontic appliance
deterioration due to the release of metallic ions, and
secondary infections.34,35 In this study, the general
unable to live for long periods in aerobic sites. This is in
levels of Candida spp decreased after T2. However, C
agreement with the current data, which showed a
albicans, the most frequently identified fungus, which is
decrease in the levels of several pathogenic species
responsible for 75% of opportunist systemic infections
(A.a.b, P nigrescens, P putida, F periodonticum, P
and has an indirect role in gingivitis, periodontitis, and
aeruginosa, P anaerobius, T dentı́cola, and T forsythia)
dental caries,36 showed decreased levels only at T3. In
over the study period. P gingivalis increased levels at
turn, L casei, P gingivalis, and P intermedia, species
T2 and T3. The levels may have altered according to
that are favored with the presence of C albicans,
the presence of oxygen and nutrients. Some species showed an increase starting from T2. These data may
are more sensitive to changes in these conditions, suggest that C albicans proliferation could trigger
which could explain the different behaviors of these imbalance in the oral environment.
periodontal pathologic species. The sample enrolled was composed of healthy
In the Streptococcus group, a significant decrease patients, with GI, PI, and GBI indices that indicated
occurred to most species at T3. This agrees with the health, and the patients’ oral hygiene was supervised
findings of previous literature, which described that and monitored monthly. Despite the absence of
these species were present in the initial stage of biofilm significant changes in the clinical indices used in the
development. They were the primary colonizers that present study, there was a difference in the microbi-
then co-aggregated with other bacteria, thus leading to ological parameters between the initial timepoint T0,
the development of a mature biofilm.23,30,31 T1, and T2, showing the important role of adequate
S mutans and S sobrinus exhibited higher levels mechanical hygiene in these patients. In this study, the
before bonding. S mutans levels decreased signifi- Hawthorne Effect might have been expected to play
cantly at T2 and showed a mild increase at T3, while S some role in motivating patients to perform better oral
sobrinus levels decreased significantly only at T3. This hygiene. At the first observational period (30 days), the
species has high virulence because of high adhesion Hawthorne Effect should have had its greatest influ-
capability, acidogenicity, and acid-uric properties. High ence, because this was a novel situation, but at that
levels of both species in a patient indicated more timepoint, an increase in the measured parameters
susceptibility to caries incidence than for patients who was observed. Patient motivation and the reinforce-
only had the presence of one species.31,32 Therefore, in ment of oral hygiene instruction resulted in oral hygiene
this study, this high risk for the incidence of caries was improvements, resulting in subsequent decreases in
observed before bonding. However, the oral hygiene the parameters evaluated. The limitations of this study,

Angle Orthodontist, Vol 89, No 1, 2019

HYGIENE PROTOCOL SUSTAINS BACTERIAL LEVELS
including the small number of enrolled subjects and the
large variability in the ages of the patients, must be
considered. Future research considering the effects of
age, longer observation periods, and additional mon-
itoring may add important new information to the
current findings.
CONCLUSION
 A dynamic change in microbial levels was found. The
decrease in the levels of complexes present was only
possible because of the mechanical method of oral
hygiene implemented in this sample.
ACKNOWLEDGMENTS
The work was supported by FAPESP, Brazil (Fundação de
Amparo à Pesquisa do Estado de São Paulo, grant 10/ 176757-
5) and CAPES for the PhD scholarship for Ana Zilda Nazar
Bergamo.
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